Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: MiR-136-3p content in cell culture medium and uptake of extracellular miR-136-3p into cultured myotubes. MiRNA content in (A) human myotubes and (B) human pancreatic islets culture media. Results were first normalized using RNU1A1 and then presented in relation to miR-23a-3p content for n = 4 different donors for myotubes cultures and n = 4 donors for human islets. Left panel (C) shows bright-field image of cultured human myotubes and right panel (C) shows a representative fluorescence image of cultured human myotubes with cells exposed to human serum-derived EVs loaded with Cy3-miR-136-3p. Cy3 fluorescence (red) is detected in the whole cytoplasm of the human myotubes. (D) Representative fluorescence image of human myotubes exposed to HEK293 culture medium with EVs loaded with Cy3-miR-136-3p (red). (E) Representative image of human myotubes exposed to EVs loaded with TexasRed-labeled with a control RNA (orange). Nuclear Hoechst staining is shown in blue. Scale bar = 100 μm. EVs = extracellular vesicles; HEK293 = human embryonic kidney; miR = microRNA; Rel = relative; RNU1A1 = U1 small nuclear RNA.

Article Snippet: Human embryonic kidney (HEK293) cells were obtained from American Type Culture Collection (ATCC) and cultured in high-glucose (4.5 g/L) Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% (vol/vol) FBS.

Techniques: Cell Culture, Fluorescence, Derivative Assay, Labeling, Control, Staining

Journal: Journal of Sport and Health Science

Article Title: Exercise training-induced extracellular miR-136-3p modulates glucose uptake and myogenesis through targeting of NRDC in human skeletal muscle

doi: 10.1016/j.jshs.2025.101091

Figure Lengend Snippet: NRDC is a direct target of miR-136-3p in human myotubes. Skeletal muscle NRDC mRNA is responsive to training and inactivity. (A) Tissue mRNA expression of NRDC from the Human Protein Atlas database showing enriched expression of NRDC in human skeletal muscle. (B) The miR-136-3p target site in the NRDC gene is highly conserved in mammals. (C) Luciferase activity in HEK293 cells co-transfected the NRDC 3’UTR and miR-136-3p with or without anti-miR136-3p inhibitors. miR-136-3p transfection downregulates NRDC (D) mRNA and (E) representative image of protein abundance in human myotubes. (F) Publicly available data ( GSE14413 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 6 weeks of endurance training ( n = 8). (G) Publicly available data ( GSE120862 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 2 months of aerobic training ( n = 10). (H) Publicly available data ( GSE14901 ) showing NRDC mRNA expression in human skeletal muscle of healthy young participants after 14 days of immobilization ( n = 24). * p < 0.05, ** p < 0.005. GSE = gene set enrichment; HEK293 = human embryonic kidney; miR = microRNA; NC = negative control; NRDC = nardilysin convertase; nTPM = normalized transcripts per million; si NRDC = small interfering RNA of NRDC ; UTR = untranslated region.

Article Snippet: Human embryonic kidney (HEK293) cells were obtained from American Type Culture Collection (ATCC) and cultured in high-glucose (4.5 g/L) Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% (vol/vol) FBS.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative Proteomics, Negative Control, Small Interfering RNA

Journal: eLife

Article Title: Modulation of human dorsal root ganglion neuron excitability by Nav1.7 inhibition

doi: 10.7554/eLife.111490

Figure Lengend Snippet: Recordings were made on a Qube 384 automated patch-clamp instrument (Sophion Bioscience) using a stable cell line with human Nav1.7 channels expressed in HEK293 cells using 10-hole recording wells. Sodium channel current was evoked by a step from –80 mV to +5 mV delivered every 3 s. Each concentration of AM-2099 was applied in 16 wells, and data from each well were accepted if the leak-corrected current at the holding voltage of –80 mV was less than 0.5 nA and the peak sodium current in control was >10 nA. Symbols show mean ± SD for wells meeting these criteria with current measured after 16 min of exposure to compound. ( A ) 12 wells for control solution containing 0.1% DMSO (0 nM), 8 for 1 nM, 8 for 3 nM, 8 for 10 nM, 8 for 30 nM, 8 for 100 nM, 12 for 300 nM, and 15 for 1000 nM. ( B ) 23 wells for control drug-free solution containing 0.1% DMSO, 12 for 1 nM, 13 for 3 nM, 15 for 10 nM, 9 for 30 nM, 11 for 100 nM, 11 for 300 nM, and 15 for 1000 nM. Fitted curves: Imax/(1+[Drug]/Kd), where Imax was fixed at the average value in the DMSO control wells (concentration of 0 nM) and the fit included weighting of data points by SD. Fit to data in (A) ignored the values from the column corresponding to 3 nM AM-2099 as it was obvious that there was an error in preparing this dilution. Internal solution (mM):140 CsF, 10 NaCl, 1 EGTA, 10 HEPES, 25 sucrose pH 7.3 w CsOH. External Solution (mM):145 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, 10 glucose, pH 7.4 w NaOH. All solutions contained 1 mg/mL Pluronic-F68. Figure 1—source data 1. Numerical data contained in .

Article Snippet: The concentration-dependence for inhibition of cloned human Nav1.7 channels by AM-2099 was determined using a Qube 384 automated patch-clamp instrument (Sophion Bioscience) using a stable cell line previously generated in the laboratory ( ) with human Nav1.7 channels (GenBank Accession Number X82835.1 ) expressed in a HEK293 cell line purchased from American Type Culture Collection (CAT# ATCC CRL-1573, ATCC, Manassas, VA).

Techniques: Automated, Patch Clamp, Stable Transfection, Concentration Assay, Control

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A–C ) Measurement of Bungarotoxin binding site (BBS) tagged Orai1-YFP in the PM of Scr (black) and STX11 shRNA-treated (red), resting (solid line), and store-depleted (broken line), HEK293 cells. Measurement of Orai1-YFP ( A ) and Alexa Fluor 647-conjugated Bungarotoxin binding ( B ) in a population of cells using flow-cytometry. ( C ) Quantification of Orai1-YFP and BTX-A647 in individual cells using confocal imaging. Line profile showing overlay of Orai1-YFP (green) and BTX-A647 (red) signal across the representative cells for individual groups. ( D ) Confocal images and quantification of Orai1-YFP and BTX-A647 in scramble and STX11 shRNA-treated, resting, and store-depleted Jurkat T cells. (n=30–40, N=3) ( E–G ) Quantification of receptor-mediated uptake and recycling in STX11 shRNA-treated cells. Scr or STX11 shRNA-treated HEK293 and Jurkat T cells were loaded with Alexa-647-Transferrin (30 min pulse), washed, chased for 0, 5, 10, and 20 min and imaged to quantify total Transferrin levels inside each cell. Representative images from HEK293 cells ( E ) and quantification from 50 to 60 HEK293 ( F ) and Jurkat T cells ( G ) is shown. N=3 ( H ) Quantification of non-receptor mediated endocytosis in STX11-depleted HEK293 cells. Representative images of FITC-conjugated Dextran uptake in Scr and STX11-shRNA-treated HEK293 cells (left panel) and quantification of the mean intensity per cell (right panel). ( I ) Quantification of biosynthesis and secretion in STX11-depleted cells. Scr or STX11 shRNA-treated HEK293 cells were transfected with secretory alkaline phosphatase (AP) expressing plasmid and levels of AP secreted into the culture supernatants were measured using p-nitrophenyl phosphate (pNPP) substrate at 48 hr post-transfection. ( J–K ) Immunolabeling of HEK293 cells to assess ER ( J ) and Golgi ( K ) health. Scr and STX11 shRNA-treated HEK293 cells were stained with anti-KDEL ( J ) and anti-GM130 antibodies ( K ), respectively, and imaged. ( L ) Measurement of ER calcium content in Scr and STX11 shRNA-treated HEK293 cells using transiently transfected ER-localized CEPIA. Cells were incubated in Ringer’s buffer containing 0 mM calcium followed by 1 mM EGTA, stimulated with 1 μM ionomycin and imaged. (N=3). Figure 2—source data 1. Folder containing the original flow cytometry files and gating strategy for . Figure 2—source data 2. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Binding Assay, shRNA, Flow Cytometry, Confocal, Imaging, Transfection, Expressing, Plasmid Preparation, Immunolabeling, Staining, Incubation

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Measurement of SOCE in STX11-depleted Jurkat and FHLH4 patient T cells. ( A–B ) Representative Fura-2 calcium imaging assay ( A ) and quantification of independent repeats ( B ) measuring thapsigargin (TG) induced SOCE in Jurkat T cells treated with Scr (black), STX11#1 (red) or STX11#2 (blue) shRNAs. (n=30–40 cells/group, N=3). ( C–D ) Representative Fura-2 calcium imaging assay ( C ) and quantification of repeats ( D ) showing reconstitution of SOCE in STX11-depleted Jurkat T cells by ectopic expression of STX11. Scr shRNA with empty vector (EV) expression (black), STX11 shRNA with EV expression (red), STX11 shRNA with STX11 expression (green). (n=30–40 cells/group, N=3). ( E ) Sanger sequencing of FHLH4 patient DNA showing deletion of a single adenine at the 752nd position and the resulting frameshift. ( F ) Schematic showing the predicted domain distribution of the wildtype versus FHLH4 mutant STX11 protein. ( G ) Western blot on WCLs of healthy donor (HD) and FHLH4 patient PBMCs showing the relative molecular weight and abundance of the wildtype and mutant STX11 bands (red arrow). ( H ) Western blot on WCLs of HEK293 cells over-expressing wildtype or FHLH4 mutant STX11 (red arrow) and empty vector. ( I ) Fura-2 calcium imaging assay measuring anti-CD3 induced SOCE in HD (black) and FHLH4 (red) T cells. (n=30–40 cells/group). ( J ) Fura-2 calcium imaging assay measuring SOCE in HD expressing empty vector (EV) (black) and FHLH4 T cells expressing human STX11 (red), respectively. (n=30–40 cells/group). ( K–L ) Representative Fura-2 calcium imaging assay ( K ) and quantification of repeats ( L ) measuring SOCE in Jurkat T cells treated either with Scr shRNA expressing empty vector (EV) (black), STX11 shRNA expressing EV (red), STX11 shRNA expressing wildtype STX11 (green), or STX11 shRNA expressing FHLH4 mutant STX11 (blue). Figure 1—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 1—source data 2. Folder containing the original sequencing result files, shown in . Figure 1—source data 3. Folder containing labeled PDF files of the original western blots for . Figure 1—source data 4. Folder containing the original images of western blots displayed in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Imaging, Expressing, shRNA, Plasmid Preparation, DNA Sequencing, Mutagenesis, Western Blot, Molecular Weight, Sequencing, Labeling

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Oligomerization of Orai1. ( A ) N-FRET analysis of Scr and STX11 shRNA-treated HEK293 cells co-expressing Orai1-CFP and Orai1-YFP before and after store-depletion. ( B–C ) Representative images ( B ) and quantification ( C ) of FRET analysis using acceptor photobleaching of Scr and STX11 shRNA-treated Orai1, Stim1 double knockdown resting HEK293 cells co-expressing Orai1-CFP (donor) and Orai1-YFP (acceptor). ( D–E ) Representative western blot ( D ) and quantification of band intensities from repeats ( E ) showing distribution of BS3 crosslinked Flag-Orai1 oligomeric bands B,C,D in control and STX11-depleted HEK293 cells. N=3. ( F ) Representative confocal images and quantification (right) of YFP-CAD localization in Scr and STX11 shRNA-treated HEK293 cells expressing Orai1-CFP. ( G–H ) Fura-2 calcium imaging assay ( G ) and quantification of repeats ( H ) to measure constitutive calcium influx in Orai1-CFP and YFP-CAD expressing, control (black) or STX11-depleted (red) HEK293 cells. n=80–90, N=3 ( I–J ) Fura-2 calcium imaging assay ( I ) and quantification of repeats ( J ) to measure constitutive calcium influx in Orai1-H134S mutant expressing control (black) and STX11-depleted (red) HEK293 cells. n=80–100, N=3 ( K–L ) Fura-2 calcium imaging assay ( K ) and quantification of repeats ( L ) to measure constitutive calcium influx in Orai1-ANSGA mutant expressing control (black) and STX11-depleted (red) HEK293 cells. n=50–60, N=3. Figure 8—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 8—source data 2. Folder containing labeled PDF files of the original western blots for . Figure 8—source data 3. Folder containing the original images of western blots shown in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Analysis, shRNA, Expressing, Knockdown, Western Blot, Control, Confocal, Imaging, Mutagenesis, Labeling, Förster Resonance Energy Transfer

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Intracellular localization of STX11 and its association with Orai1. ( A ) (Upper panel) Schematic showing epitopes for anti-STX11 and anti-HA antibodies used in the lower panel. (Lower panel) Representative confocal images of HEK293 cells transfected with either HA-tagged STX11 or untagged STX11 and stained using anti-HA antibody or anti-STX11 antibody, respectively. N=3 ( B–D ) Localization of HA-tagged STX11 with respect to Orai1-YFP in the basal as well as middle plane of resting ( B ) and store-depleted ( C ) HEK293 cells. ( D ) Pearson’s correlation coefficient showing Orai1 and STX11 colocalization from ( B ) and ( C ). N=3. ( E ) Representative confocal images of FHLH4 mutant STX11 expressing HEK293 cells. ( F–G ) Co-IP to assess STX11 binding to Orai1. Whole cell lysates of resting and store-depleted HEK293 cells co-expressing either Flag-Orai1 and STX11 ( F ) or Orai1-Myc and STX11 ( G ) were subjected to IP and western blot using anti-Myc, anti-Flag, or anti-STX11 antibodies, as indicated. (N=3) ( H ) Schematic showing key domains of STX11 and Orai1 used for in vitro pull-down assays. ( I ) Pull-down assay showing in vitro binding of His-tagged STX11 to Orai1. (Top) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. Input: 1/5 th of the protein. N=4. ( J ) Pull-down assay showing in vitro binding of His-tagged H abc domain of STX11 to Orai1 fragments. (Top) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-His antibody. Input: 1/5 th of the protein. N=3. ( K ) Binding free energy distribution of STX11-H abc and Orai1 C-terminus interactions. ( L ) Sphere and cartoon representation of the structure of STX11 H abc (green) and Orai1 C-terminus (cyan) complex after MD simulation. The N-termini are highlighted in blue, and C-termini in red. ( M ) Representative confocal images showing localization of STX11 with respect to Orai1:Stim1 puncta in store-depleted cells. HEK293 cells expressing Orai1-YFP, CFP-Stim1 were transfected with HA-tagged STX11, store-depleted, and stained using anti-HA antibody. (N=3) ( N ) Pearson’s correlation coefficient showing quantification of the localization of STX11 with respect to Stim1:Orai1 puncta in store-depleted cells from ( M ). Figure 5—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 5—source data 2. Folder containing labeled PDF files of the original western blots for indicating the relevant bands. Figure 5—source data 3. Folder containing the original images of western blots shown in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Confocal, Transfection, Staining, Mutagenesis, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, In Vitro, Pull Down Assay, Labeling

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A ) Schematic showing the design of recombinant Orai1-BBS-YFP fusion protein. Shown here is the placement of the bungarotoxin binding site (BBS) inside the second extracellular loop and YFP tag in the C-terminus of Orai1 as well as binding of BTX-A647 to BBS. Created with BioRender.com . ( B ) Representative quantitative PCR to assess the efficiency of knockdown in shRNA-treated HEK293 cells stably expressing Orai1-BBS-YFP. Total RNA extracted from cells treated with STX11 shRNA was subjected to qPCR analysis using Taqman probes for STX11. Data were normalized to RPL30 housekeeping control. ( C ) Representative Fura-2 calcium imaging assay measuring Thapsigargin (TG) induced SOCE in Orai1-BBS-YFP expressing HEK293 cells treated with Scr or STX11 shRNA. ( D ) Representative quantitative PCR to assess the efficiency of knockdown in shRNA-treated Jurkat T cells expressing Orai1-BBS-YFP. Total RNA extracted from cells treated with STX11 shRNA was subjected to qPCR analysis using Taqman probes for STX11. Data were normalized to RPL30 housekeeping control. Figure 2—figure supplement 1—source data 1. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Recombinant, Binding Assay, Real-time Polymerase Chain Reaction, Knockdown, shRNA, Stable Transfection, Expressing, Analysis, Control, Imaging, Clinical Proteomics

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: Analysis of STX11 and Orai1 mutants. ( A ) Schematic showing STX11 mutations (Top). STX11 N147A_E150A mutant expressing HEK293 co-labeled with Alexa Fluor-488 conjugated wheat germ agglutinin (WGA) or co-transfected with Orai1-YFP and immunolabeled with anti-STX11 to assess co-expression in PM of resting and store-depleted cells (Bottom). Scale bar 10 μm. ( B ) Western blot to compare total expression of mutant and wildtype STX11 using anti-STX11 antibody. ( C–D ) Representative Fura-2 calcium imaging assay ( C ) and quantification of repeats ( D ) measuring rescue of SOCE in Jurkat cells treated either with Scr shRNA (black) or STX11 shRNA expressing empty vector (red), wildtype STX11 (green), or N147A_E150A mutant STX11 (blue). ( E ) Coomassie blue stained SDS-PAGE showing expression and purification of His-tagged wildtype or N147A_E150A mutant STX11 (red arrow) from E. coli . ( F–G ) Pull-down assay measuring their binding to MBP-tagged Orai1 C-terminus. (Top) Ponceau S staining showing the input of MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. N=4 ( G ) Quantification of STX11 band intensities in ( F ). ( H ) Schematic showing Orai1 mutations (Top). Confocal images of HEK293 cells expressing YFP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 (Bottom). ( I–J ) Representative Fura-2 calcium imaging assay ( I ) and quantification of repeats ( J ) measuring rescue of SOCE in HEK293 cells treated either with Scr (black) or Orai1 shRNA expressing empty vector (red), wildtype Orai1 (green), or R289A_E272A_E275A_E278A mutant Orai1 (blue). ( K–L ) Pull-down assay measuring the binding of MBP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 C-terminus to wildtype or N147A_E150A mutant STX11. (Top) Ponceau S staining showing the input of MBP-tagged Orai1 fragments. (Bottom) Western blot using anti-STX11 antibody. N=4. ( L ) Quantification of STX11 band intensities in ( K ). ( M–N ) Schematic showing CAD domain in Stim1 (top panel) and representative confocal images (lower panel) ( M ) and quantification ( N ) of PM localized YFP-CAD in CFP-tagged wildtype or R289A_E272A_E275A_E278A mutant Orai1 expressing HEK293 cells. ( O–P ) Representative Fura-2 calcium imaging assay ( O ) and quantification of repeats ( P ) of constitutive calcium flux from cells imaged in panels ( M–N ). Figure 6—source data 1. Folder containing labeled PDF files of the original gels and western blots for . Figure 6—source data 2. Folder containing the original images of gels and western blots shown in . Figure 6—source data 3. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Analysis, Mutagenesis, Expressing, Labeling, Transfection, Immunolabeling, Western Blot, Imaging, shRNA, Plasmid Preparation, Staining, SDS Page, Purification, Pull Down Assay, Binding Assay, Confocal

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A–C ) Fura-2 calcium imaging assay measuring SOCE in cells treated with Scr shRNA or different shRNA sequences against SNAP23 ( A ) SNAP25 ( B ) SNAP29 ( C ). Average traces from 30 to 40 cells per group are shown. ( D ) Western blot images of co-IPs to test the association of Orai1 with SNAP23/SNAP25/SNAP29. HEK293 cells were co-transfected with Orai1-Myc and untagged SNAP23/SNAP25/SNAP29, store-depleted, lysed, and subjected to co-IP followed by western blot as indicated. (N=2) ( E ) Western blot images of co-IP to test the association of Stim1 with SNAP23/SNAP25/SNAP29. HEK293 cells were co-transfected with YFP-Stim1 and Myc-tagged SNAP23/SNAP25/SNAP29, store-depleted, lysed, and subjected to co-IP followed by western blot as indicated. (N=2). Figure 2—figure supplement 3—source data 1. Numerical data underlying the quantitative analyses represented in . Figure 2—figure supplement 3—source data 2. Folder containing labeled PDF files of the original western blots for . Figure 2—figure supplement 3—source data 3. Folder containing the original images of western blots shown in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Imaging, shRNA, Western Blot, Transfection, Co-Immunoprecipitation Assay, Labeling

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (co-IP) to test the association of Stim1 with STX11. HEK293 cells were co-transfected with Stim1-Myc-His and STX11, store-depleted, lysed, and subjected to co-IP followed by western blot as indicated. (N=2) ( B ) Quantification of the fraction of Orai1 inside and outside Stim1:Orai1 clusters in store-depleted HEK293 cells expressing CFP-Orai1 and Stim1-YFP. Figure 5—figure supplement 4—source data 1. Folder containing labeled PDF files of the original western blots for . Figure 5—figure supplement 4—source data 2. Folder containing the original images of western blots shown in . Figure 5—figure supplement 4—source data 3. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Expressing, Labeling

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A–L ) Quantification of Orai1 and Stim1 intensities in the ER-PM puncta of store-depleted cells. ( A–B ) Representative images of resting ( A ) and store-depleted ( B ) shRNA-treated HEK293 cells expressing N-terminal CFP-tagged Orai1 (CFP-Orai1) and C-terminal YFP-tagged Stim1 (Stim1-YFP) from confocal microscopy. ( C–F ) Mean Stim1-YFP intensity ( C ) and area ( D ) of Stim1 clusters. ( E ) Fraction of CFP-Orai1 intensities inside Stim1-YFP clusters of shRNA-treated cells, post store-depletion. ( F ) Pearson’s correlation coefficient of Stim1 and Orai1 co-localization inside Stim1 puncta. N=3. ( G–L ) Quantification of Orai1 and Stim1 intensities in the ER-PM puncta of C-terminal YFP-tagged Orai1 (Orai1-YFP) and N-terminal CFP-tagged Stim1 (CFP-Stim1) expressed in HEK293 cells and imaged using TIRF microscopy. ( G ) Representative TIRF images of resting and store-depleted shRNA-treated HEK293 cells. Scale bar 10 μM. ( H–K ) Mean CFP-Stim1 intensity ( H ) and area ( I ) of Stim1 clusters. Fraction of Orai1-YFP intensities ( J ) and area ( K ) of Orai1 clusters in control and STX11-depleted cells. ( L ) Pearson’s correlation coefficient of Stim1 and Orai1 co-localization inside Stim1 puncta. N=2. ( M–N ) Representative Fura-2 calcium imaging assay ( M ) and quantification of repeats ( N ) measuring TG-induced SOCE in Orai1-YFP and CFP-Stim1 expressing HEK293 cells treated with Scr (black) or STX11 (red) shRNA. ( O–Q ) Representative TIRF images of GFP-MAPPER in store-depleted Scr and STX11 shRNA-treated HEK293 cells ( O ). Quantification of area ( P ) and intensity ( Q ) of GFP-MAPPER puncta. n=15. Figure 7—source data 1. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: shRNA, Expressing, Confocal, Microscopy, Control, Imaging

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A–H ) Fura-2 calcium imaging assays measuring thapsigargin (TG) induced SOCE in HEK293 and/or Jurkat cell lines treated with Scr shRNA or different shRNA sequences against the gene of interest for 3–4 days. Shown here are the average traces from 30 to 40 cells per group. Figure 1—figure supplement 1—source data 1. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Imaging, shRNA

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A–B ) Representative Fura-2 calcium imaging assay ( A ) and its quantification from repeats ( B ) measuring thapsigargin (TG) induced SOCE in HEK293 cells treated with Scr or STX11 shRNA. Bars show relative SOCE ± SE from three independent experiments in ( A ) where SOCE from Scr shRNA-treated group in one experiment was set at 100%. ( C ) Quantitative PCR to assess the efficiency of knockdown in shRNA-treated HEK293 ( C ) and Jurkat T cells ( D ) from three independent repeats. Total RNA extracted from cells treated with STX11 shRNA was subjected to qPCR analysis using Taqman probes for STX11. Data were normalized to beta-actin housekeeping control. Figure 1—figure supplement 2—source data 1. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Imaging, shRNA, Real-time Polymerase Chain Reaction, Knockdown, Analysis, Control

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A ) Schematic showing a comparison of the key domains in STX11 and STX1A. ( B ) Western blot of whole cell lysates prepared from HEK293 cells expressing YFP-STX11 and STX11-YFP. Note the mismatch in molecular weight of the N- versus C-terminally tagged STX11 and the degradation products (arrows). N=3. ( C ) Confocal images showing localization of ectopically expressed mCherry-STX11 or STX11-mCherry in HEK293 cells. N=3. ( D ) Pull-down assay showing in vitro binding of His-tagged SNARE domain of STX11 to MBP-tagged Orai1 N- and C-termini. (Top panel) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 cytosolic tails. (Bottom panel) Western blot using anti-His antibody. Figure 5—figure supplement 1—source data 1. Folder containing labeled PDF files of the original western blots for . Figure 5—figure supplement 1—source data 2. Folder containing the original images of western blots shown in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Comparison, Western Blot, Expressing, Molecular Weight, Confocal, Pull Down Assay, In Vitro, Binding Assay, Staining, Labeling

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A–D ) Representative confocal images ( A ) and quantification ( B–D ) of C-terminally YFP-tagged wild-type or R289A_E272A_E275A_E278A mutant Orai1 and CFP-Stim1 intensities and area of clusters in store-depleted HEK293 cells. n=20. Figure 7—figure supplement 1—source data 1. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Confocal, Mutagenesis

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A ) Schematic representation of SOAR domain in full-length Stim1. ( B ) Cartoon showing the design of the Orai1-SOAR-SOAR-EGFP (Orai1-S-S-GFP) construct. Created with BioRender.com . ( C ) Representative Fura-2 calcium imaging assay to measure constitutive calcium influx in Orai1-S-S-GFP expressing control (black) or STX11-depleted (red) HEK293 cells. Cells were imaged in Ringer’s buffer containing 0 mM followed by 2 mM extracellular Ca 2+ . ( D ) Quantification of constitutive calcium influx across experiments as shown in ( C ). n=90–100, N=3. Figure 8—figure supplement 2—source data 1. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Construct, Imaging, Expressing, Control, Clinical Proteomics, shRNA

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A ) Schematic representation of H134S mutation in full-length Orai1. ( B ) Quantification showing percent Orai1-H134S-YFP intensity normalized to Orai1-WT-YFP in the PM of Scr and STX11 shRNA-treated HEK293 cells. n=30–40. ( C ) Schematic representation of ANSGA mutation in full-length Orai1. ( D ) Quantification showing percent Orai1-ANSGA-YFP intensity normalized to Orai1-WT-YFP in the PM of Scr and STX11 shRNA-treated HEK293 cells. n=30–40. Figure 8—figure supplement 3—source data 1. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Mutagenesis, shRNA

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: ( A–F ) Fura-2 calcium imaging assays measuring thapsigargin (TG) induced SOCE in HEK293 or Jurkat T cells treated with Scr shRNA or different shRNA sequences against the gene of interest for 3–4 days. Shown here are the average traces from 30 to 40 cells per group. Figure 1—figure supplement 3—source data 1. Numerical data underlying the quantitative analyses represented in .

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: Imaging, shRNA

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques:

Journal: eLife

Article Title: Syntaxin11 deficiency inhibits CRAC channel priming to suppress cytotoxicity and gene expression in T lymphocytes independent of membrane trafficking

doi: 10.7554/eLife.111335

Figure Lengend Snippet: (C-D) Quantification of individual repeats of Scr and STX11 shRNA-treated HEK293 cells expressing Orai1-H134S (C) and Orai1-ANSGA (D) mutants.

Article Snippet: Cell line ( Homo sapiens ) , HEK293 , ATCC , CRL-1573 (RRID: CVCL_0045 ) , , , — and associated figure supplements .

Techniques: shRNA, Expressing

Journal: Nature Communications

Article Title: Multimodal substrate recruitment enables CTLH MKLN1 E3 ligase to target acetylated N-, C-, and internal degrons

doi: 10.1038/s41467-026-78311-4

Figure Lengend Snippet: a Cartoon depicting the E3-MKLN1–Sub-MKLN1 tetrameric assembly, highlighting the discoidin and Kelch domains, and Asn447 within Kelch domain Loop-1 (starred), which is central to the E3-MKLN1–Sub-MKLN1 interface. b Cartoon depicting the Kelch domain central channel, which recognizes the C-terminal Arg of ZMYND19 and FAM72A, highlighting contact residues Gly273, Trp291, and Tyr346 (starred). c In vitro ubiquitylation assay comparing CTLH MKLN1 WT and the N447R mutant toward MKLN1, ZMYND19, and FAM72A-UNG2, n = 3 technical replicates. d Immunoblot analysis of MKLN1 levels in MKLN1-deficient cells transiently expressing WT, W291A/G273W, or N447R 3×Flag-MKLN1 , monitoring Torin1-dependent degradation after 16 h, Torin1 + MG132 (16 h) co-treatment, or matched DMSO controls. Actin serves as a loading control. e Quantification of MKLN1 immunoblots from ( d ). Data from each well were normalized to actin, and then each set expressing a particular 3×Flag-MKLN1 construct was normalized to the DMSO control for that construct. The graph shows results of mean ± s.d. of n = 4 biological replicates. P -values were calculated using two-way ANOVA with Dunnett’s multiple comparison tests ( p -value significance <0.05). f Top: sequence alignment of MKLN1, ZMYND19, and FAM72A C-termini. Bottom: superposition of cryo-EM structures of FAM72A (blue) and ZMYND19 (magenta) C-termini bound within the E3-MKLN1 Kelch domain central channel, highlighting their similar contacts between MKLN1 Gly273, Trp291, Tyr346, Arg407, and Arg517, and the terminal Arg residues of FAM72A (Arg149) and ZMYND19 (Arg227). g In vitro ubiquitylation assay examining effect of W291A/G273W Kelch central channel mutant on CTLH MKLN1 activity toward MKLN1, ZMYND19 and FAM72A-UNG2, n = 3 technical replicates. h Co-immunoprecipitation (using anti-RANBP9 antibody) in MKLN1- and ZMYND19-deficient HEK293 cells transiently expressing HA-ZMYND19 and 3×Flag-MKLN1 (WT or indicated N447R, or W291A/G273W mutants), assessing the role of Kelch channel surfaces on ZMYND19 association with the CTLH complex. Immunoprecipitates and inputs are shown. Actin serves as a loading control ( n = 2 biological replicates). i Immunoblot analysis of UNG and FAM72A levels in MKLN1 −/− knockout HEK293 cells transiently co-transfected with constructs expressing FAM72A and 3×Flag-MKLN1 WT or indicated mutant versions (W291A/G273W, or N447R) following mock (DMSO) treatment or treatment with proteasome inhibitor MG132. j Quantification of UNG2 immunoblots from ( i ). Data from each well were normalized to actin, and then to the DMSO control for cells transfected with a construct expressing WT 3×Flag-MKLN1 . The graph shows results of mean ± s.d. of n = 3 biological replicates. P -values were calculated using two-way ANOVA with Dunnett’s multiple comparison tests ( p -value significance <0.1). k Fluorescence polarization assay measuring binding of fluorescein-labeled peptides corresponding to the C-terminal 19 residues of ZMYND19 or FAM72A to the various versions of the MKLN1 Kelch domain (WT, W291A/G273W, or W291A/Y346A). K D for the ZMYND19 peptide–WT MKLN1 interaction is indicated. Data shown are the mean ± s.d. ( n = 3 technical replicates). l Co-immunoprecipitation via anti-RANBP9 antibody or rabbit IgG as a negative control in parental and ZMYND19 −/− knockout HEK293 cells transfected with constructs expressing HA-ZMYND19 (WT, ΔC-ter 3 res, or ΔC-ter Arg), assessing the role of the ZMYND19 C-terminal region in association with the CTLH complex. Immunoprecipitates and inputs are shown. Actin serves as a loading control ( n = 5 biological replicates). m Immunoblot analysis of ZMYND19 and MKLN1 levels in ZMYND19 −/− knockout HEK293 cells transiently expressing HA-ZMYND19 WT or ΔC-ter Arg mutant, and treated either with DMSO as control, Torin1, or Torin1 and proteasome inhibitor MG132. Actin serves as a loading control. n Quantification of ZMYND19 immunoblots from ( m ). Data from each well were normalized to actin, and then each set transfected with a particular HA-ZMYND19 expression construct was normalized to the DMSO control for that construct. The graph shows results of mean ± s.d. of n = 5 biological replicates. P -values were calculated using two-way ANOVA with Dunnett’s multiple comparison tests ( p -value significance <0.05). In vitro ubiquitylation assays examining the effect of C-terminal mutations in ZMYND19 ( o ) or FAM72A ( p ) on ubiquitylation by CTLH MKLN1 . n = 2 technical replicates. Source data are provided as a file.

Article Snippet: Flp-In T-Rex HEK293 cells (ATCC CRL-1573) were purchased from Thermo Fisher Scientific (catalog no. R78007 ).

Techniques: In Vitro, Ubiquitin Assay, Mutagenesis, Western Blot, Analysis, Expressing, Control, Construct, Comparison, Sequencing, Cryo-Electron Microscopy, Activity Assay, Co-Immunoprecipitation Assay, Knock-Out, Transfection, Fluorescence, Binding Assay, Labeling, Negative Control

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet: Adaptable expression ratios of the creatine transporters CRT1 and MCT12 in HEK293 SLC6A8 −/− cells (A) Scheme of the CRT1 3xHA -P2A-K n -MCT12 2xmyc construct. (B) Immunoblot analysis of CRT1 3xHA -P2A-K n -MCT12 2xmyc . (C) Ratio of MCT12 2xmyc and CRT1 3xHA expression as determined by immunoblotting ( n = 5). Data are represented as mean ± SEM. (D) Immunoblot analysis of CRT1 3xHA -P2A-K n -MCT12 2xmyc . (E) Ratio of MCT12 2xmyc and CRT1 3xHA expression as determined by immunoblotting ( n = 4). Data are represented as mean ± SEM. See also .

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Expressing, Construct, Western Blot, Analysis

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet: Relative cell surface expression of the creatine transporters CRT1 and MCT12 in HEK293 SLC6A8 −/− cells (A) Scheme of the CRT1 eGFP -P2A-K 0 -MCT12 mCherry (a) and MCT12 mCherry -P2A-K 0 -CRT1 eGFP construct (b). (B) Ratio of mCherry and eGFP expression in eGFP-P2A-K 0 -mCherry (K 0 , as a control), CRT1 eGFP -P2A-K 0 -MCT12 mCherry , and MCT12 mCherry -P2A-K 0 -CRT1 eGFP constructs as determined by flow cytometry ( n = 6). Data are represented as mean ± SEM. (C) Live-cell confocal microscopy of HEK293 SLC6A8 −/− cells expressing CRT1 eGFP -P2A-K 0 -MCT12 mCherry and MCT12 mCherry -P2A-K 0 -CRT1 eGFP . The plasma membrane was stained with CellMask Deep Red. Scale bars, 20 μm. (D) Analysis of cell surface expression of CRT1 eGFP and MCT12 mCherry . n = 30 (CRT1 eGFP -P2A-K 0 -MCT12 mCherry ); n = 28 (MCT12 mCherry -P2A-K 0 -CRT1 eGFP ).

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Expressing, Construct, Control, Flow Cytometry, Confocal, Microscopy, Clinical Proteomics, Membrane, Staining, Analysis

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet:

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Cell-Signaling, Virus, Recombinant, Clinical Proteomics, Membrane, Staining, Cloning, Software